BIOCHEMICAL EVIDENCE FOR THE PRESENCE OF LYSOSOMES IN THE EPIDERMIS*

Transcription

1 ThE JOURNAL OP INVESTIGATiVE DERMATOLOGY Copyright 1966 by The Williams & Wilkins Co. Vol. 47, No. 5 Printed in U.S.A. BIOCHEMICAL EVIDENCE FOR THE PRESENCE OF LYSOSOMES IN THE EPIDERMIS* CHARLES H. DICKEN, M.D. AND RICHARD H. DECKER, PH.D Reviews (1, 2) of studies on lysosomes suggest that these organelles may play an important role in normal processes and in diverse pathologic conditions. Since lysosomes of different tissues may vary to a considerable extent, it is important to identify and characterize these organelles in the particular tissue to be rnvestigated. The presently available evidence for the presence of lysosomes in the epidermis is morphologic or histochemical. In electron micrographs, subcellular structures which resemble lysosomes have been seen in keratinocytes (3), in Langerhans cells (4, 5), and in intraepiderma! eccrine duct cells (6). Subeellular particles containing acid phosphatase have also been demonstrated in electron micrographs of keratinocytes of normal (7) and abnormal epidermis (8). Diengdoh (9) reported latency (time-related appearance) of acid phosphatase in mouse epidermis by using a histochemical technique and the light microscope. However, Eisen and coworkers (10) were unable to confirm this in human epidermis. Morphologic and histochemical methods cannot fulfil all the criteria for lysosomes (1). We have used biochemical methods for the demonstration of lysosomes in the epidermis of man and of the hairless mouse. METHODS Human skin was obtained from the abdomen at autopsy or from surgical amputations. Skin from the backs of hairless mice was also used. Epidermis was removed by the stretch method (11) and was immediately chilled in ice-cold, 0.3M sucrose buffered with 0.O1M phosphate buffer, ph 7.3. The tissue was homogenized in small batches for 10-second intervals at one-third to one-half speed in a VirTis Model 23 with the micro attachment; the Mayo Clinic and Mayo Foundation, Rochester, Minnesota. This investigation was supported in part by the Kieckhefer Research Fund and by Research Grant 2A-5299 from the National Institutes of Health, Pubhc Health Service. Presented at the Twenty-seventh Annual Meeting of The Society for Investigative Dermatology, Inc., Chicago, Illinois, June 26, the homogenate was filtered through one or two layers of cheesecloth to remove gross tissue fragments. The cloudy suspension was separated into three fractions, designated "nuclear," "lysosomal," and "supernatant" fractions, by the procedure of differential centrifugation indicated in Table I. Acid phosphatase was determined fluorometrically by adapting the procedure of Campbell and Moss (12) to the Turner Model 111 fluorometer with primary filter no (360 me) and secondary filter no. 47B (415 mae). Acid phosphatase activity was used to determine latency and specific activity. p-glucuronidase was assayed by a modification of the method of Fishman and coworkers (13), aryl sulfatase was determined by the method of Roy (14), and cathepsin was determined by the method of Gianetto and de Duve (15); these enzymes were measured only on homogenates because of the relative insensitivity of the assay procedures for their detection. Two technics were used to study the latent properties of the isolated lysosomes. According to the method of de Duve and co-workers (16), the lysosome suspension was incubated with substrate with or without exposure to disrupting conditions (for example, Triton-X-100). Thus, the access of substrate to enzymes still associated with granules is measured. This assay necessitates short incubation times since the ph of assay is sufficient to cause disruption of lysosomes. The acid phosphatase assay was the only procedure sensitive enough for this technic. By following the alternate method of Dingle (17), the particles were removed by centrifugation after pre-incubation with disrupting agents. The enzyme activity found in the superliate measures the release of enzyme from the granules caused by the test conditions. Protein was determined in the fractions by the method of Lowry and co-workers (18). Enzyme activities of the three fractions were measured after treatment with 0,1% Triton-X-100 for calculation of the specific and total activities because this agent increased the activity in the nuclear and lysosomal fractions. RESULTS We were able to demonstrate the activity of four of the enzymes associated with lysosomes in homogenates of the epidermis from man and from the hairless mouse. These enzymes are shown in Table II along with other enzymes that are probably associated with lysosomes and that have been demonstrated by biochemical or histochemical methods to be in epidermis. Acid phosphatase was assayed to determine the die-

2 LYSOSOMES IN EPIDERMIS 427 TAI3LE I Fractionation procedure Homogenate (0.3M sucrose in 0.O1M phosphate buffer) Filter through cheese cloth Filtrate Residu discarded Centrifuged (700 X g, 10 minutes) Nuclear fraction Supernatan fraction Centrifuged (12,000 X g, 10 minutes) Lysosomal fraction Supernatan1 fraction Resuspended in 0.25M sucrose Centrifuged (12,000 X g, 10 minutes) Supernat discarded tribution of activity and latency of the fractions. Although lysosomal acid phosphatase is usually measured with /3-glycerol phosphate as substrate, we employed a-naphthyl phosphate in a fluorometric assay method in order to obtain the sensitivity necessary for these studies. We found that the activity measured by this method was lysosomal acid phosphatase because it was sedimentable, showed latent activity, and had the same distribution as /3-glucuronidase. The distribution of acid phosphatase activity among the nuclear, lysosomal, and supernatant fractions is shown in Table III. Although the lysosomal fraction contained a relatively small part of the total activity, it also contained a smaller percentage of the total protein. That the fractionation procedure resulted in a purification of lysosomes is indicated by the increase in specific activity (enzyme activity per amount of protein) of the lysosomal fraction compared to the other fractions (Fig. 1). The relative specific activity of the iysosomal fraction could usually be increased, but Lysosomal fraction resuspended in 0.25M sucrose TABLE II Some lysosomal hydrotases found in epidermis Enzymes found, this study Other enzymes reported in literature Acid phosphatase -Glucuronidase Aryl sulfatase Cathepsin TABLE III Distribution of acid phosphatase activity in )ractions of epidermal homogenate Fraction Nuclear Supernatant Lysosomal Nonspecific esterases (19) Lipases (19) Ribonuclease (20) Deoxyribonuelease (20) Per cent of total activity (mean) Hairless mouse Human

3 428 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY this generally occurred at the expense of the total activity of the fraction. Latency of the lysosomal fraction was demonstrated most effectively with Triton-X-100 g,.- $ Human epidermis Fm. 1. Relative specific activity of acid phosphatase in fractions of homogenates of epidermis from man and from hairless mouse. N = nuclear; L lysosomal; S = supernatant. and the centrifugation technic of Dingle (17) (Fig. 2 right). With mouse preparations, Tnton-X-100 caused a fivefold increase in activity compared with a control; sodium cholate caused a 2.7-fold increase (unpublished data). Hypotonic conditions were less effective, while freezing and thawing and acidic ph were without effect. Acid ph and hypotonic conditions caused only modest stimulation compared with that induced by Triton-X-100 when latency was determined by the procedure of de Duve and co-workers (16) (Fig. 2 left). Results with human skin (Fig. 3) were nearly identical to those obtained with mouse epidermis. COMMENT Four biochemical criteria for establishing the presence of lysosomes have been fulfilled for the epidermis from man and from the hairless mouse: (1) epidermal preparations contained at least eight acid hydrolases which are known to be associated with lysosomes; (2) a portion of one of the enzymes, acid phosphatase, was lincentrifuged Centrifuged nrill 10 - HnnnhI 0 I C Ff1 ph H NaF T C F+T p1-i H NaF Fm. 2. Demonstration of latency of acid phosphatase in lysosomal fraction from epidermis of hairless mouse. Abbreviations: T = 0.1% Triton-X-100; C = control; F + T freezing and thawing xlo; ph ph5; H = hypotonic conditions (distilled water); NaF.O1M sodium fluoride. Left, Method of de Duve and co-workers (16). Right, Method of Dingle (17).

4 LYSOSOMES IN EPIDERMIS 429 Fia. 3. Demonstration of latency of acid phosphatase in lysosomal fraction from human epidermis. Abbreviations and methods are the same as in Figure 2. sedimented by centrifugation under conditions which, with liver, yield a fraction known to contain lysosomes; (3) the acid phosphatase in the sedimented fraction possessed the highest specific activity of all fractions containing the enzyrne; and (4) the fraction contained latent acid phosphatase activity, demonstrable with Triton-X-100. Morphologic studies indicate that the lysosomal content of the epidermis is not dependent on one cell type. The keratinocyte (3, 7, 8), intraepidermal eccrine duct cell (6), and Langerhans cell (4, 5) all probably contribute to the epidermal lysosome content. The extent to which each contributes to the total would be expected to vary with cell population and with rate of metabolism of each cell type. The distribution of activity (Table III) depended to some extent on the technic employed during homogenization, quite likely because the force required to break the cells also breaks many lysosonies. Human skin was difficult to homogenize, and the yield of lysosomal fraction was low. The homogenization of mouse epidermis was accomplished more easily, and the higher yield of enzyme activity in the lysosomal fraction reflects this. Histochemically, acid phosphatase is found in significant amounts in the stratum granulosum and upper stratum corneum. The acid phosphatase activity in this location is not membrane limited. The high enzyme activity in a wash of the scraped-off epidermis before any homogenization suggests that this may be a significant factor in the high enzyme activity found in the supernatant fraction. The amount of activity that would be sedimented with the microsomal fraction was not determined, but it would be of interest in view of the finding of significant amounts of enzyme activity in this fraction in liver (21). The breaking of the lipoprotein membrane of the lysosome is usually associated with release or solubilization of the hydrolytic enzymes. The method of de Duve and co-workers (16) does not necessarily measure the solubilization of the enzyme(s) because the lysosomes are not centrifuged after releasing

5 430 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY agents have been used. In this method intact lysosomes are used as a control and assays are short (10 minutes). In this manner, increased activity of the lysosomal enzymes in those samples previously treated with disrupting agents is demonstrated compared to the untreated lysosome samples. If the disrupting agents are used on the lysosomes and the solution is then centrifuged, the enzymic activity released into the supernate is less than the enzyme activity of a similarly treated duplicate that is not centrifuged. This is usually explained by stating that the difference is due to absorption of enzyme(s) on the walls of the disrupted lysocomes and that the fragments are then sedimentable. Another explanation or extension to the lysosome concept was suggested by Koenig (22): that the internal matrix of the lysosome is composed of macromolecules of enzyme and nonenzyme protein conjugated to acidic glycolipids by ionic, covalent, or other bonds. Electron micrographs of lysosornes do indicate that an internal structure exists (8, 23), and Koenig and Jibril (24) showed release of lysosomal enzymes with diverse cationic molecules. Acid ph, hypotonic solutions, and freezing and thawing readily release liver lysosomal enzymes (1). This means that these agents must disrupt the membrane and the internal matrix. We found little or no solubiization of enyme with acid ph, hypotonic solutions, or freezing and thawing, but an epidermal lysosome solution at ph 5 showed 50% of the activity of an epidermal lysosome solution made with 0.1% Triton-X-100. This finding suggests that the membrane may be broken by the above agents but that the internal matrix or large pieces of it continue to bind the enzyme and that it is therefore still sedimentable although 50% of the enzyme has been activated by the lowered ph. The increased activity and solubilization found with Triton-X-100 would be due to disruption of the membrane and the internal matrix. The process of keratinization is in many ways similar to cell death or autolysis. As epidermal cells progress from the basal layer to the stratum corneum, there is a disappearance of such organelles as the mitochondria and the nucleus. It would appear then that lysosomes may play a role in the degradative processes associated with formation of the stratum corneum. Lysosomes have the enzymes capable of this function, such as deoxyribonuclease, ribonuclease, protease, esterase, and lipase, and the acid ph in the upper layers of the epidermis is optimal for these acid hydrolases. The lysosomes tentatively appear to be implicated in tissue injury and inflammation, immune response, connective tissue disease, mitosis, fever, metabolic diseases, photosensitivity, and specific organ diseases (2, 25). The present evidence is circumstantial for lysosomal involvement in all of these processes, except in the case of some storage diseases, in which specific lack or diminution of a lysosomal enzyme has been shown (26 28). The role of lysosomes in skin diseases should be investigated, especially in view of their reported labiization or stabilization by such agents as ultraviolet irradiation, phylloerythrin, cantharidin, vitamin A, hydrocortisone, chioroquine, and antihistamines. SUMMARY Four biochemical tests have demonstrated the presence of lysosomes in the epidermis of man and of the hairless mouse: (1) presence of acid hydrolases, (2) sedimentability, (3) increased specific activity, (4) structure-linked latency. Epidermal homogenates from man and from the hairless mouse have been shown to contain four lysosomal enzymes: acid phosphatase, cathepsin, /3-glucuronidase, and aryl sulfatase. Acid phosphatase activity was used to demonstrate sedimentability of the lysosomal fraction and structure-linked latency. Acid ph, hypotonic solution, and freezing and thawing did not solubilize epidermal lysosomes. Triton- X-100 produced time-dependent solubiization (latency) of epidermal lysosomes by two methods. REFERENCES 1. De Duve, Christian: The lysosome concept. In De Reuck, A. V. S. and Cameron, Margaret P.: Lysosomes, pp Boston, Mass., Little, Brown & Company, Weissmann, Gerald: Lysosomes. New Eng. J. Med., 73: 1084; 1143, Mishima, Y.: Macromolecular changes in pigmentary disorders. Arch. Derm. (Chicago), 91: 519, Breathnach, A. S.: Observations on cytoplasmic organelles in Langerhans cells of human epidermis. J. Anat., 98: 265, 1964.

6 LYSOSOMES IN EPIDERMIS Zelickson, A. S.: The Langerhans cell. J. Invest. Derm., 44: 201, Hashimoto, Ken, Gross, B. G. and Lever, W. F.: The ultrastructure of the skin of human embryos. I. The intraepidermal cccrine sweat duct. J. Invest. Derm., 45: 139, Olson, R. L. and Nordquist, R. E.: Ultramicroscopic localization of acid phosphatase in human epidermis. J. Invest. Derm., 46: 431, Prose, P. H., Sedlis, Emilia and Bigelow, Monica: The demonstration of lysosomes in the diseased skin of infants with infantile eczema. J. Invest. Derm., 45: 448, Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, Eisen, A. Z., Arndt, K. A. and Clark, W. H., Jr.: The ultrastructural localization of acid phosphatase in human epidermis. J. Invest. Derm., 43: 319, Van Scott, E. J.: Mechanical separation of epidermis from the corium. J. Invest. Derm., 18: 377, Campbell, Diana M. and Moss, D. W.: Spectrofluorimetric determination of acid phosphatase activity. Clin. Chem. Acta, 6: 307, Fishman, W. H., Springer, B. and Brunetti, R.: Application of an improved glucuronidase assay method to the study of human blood p-glucuronidase. J. Biol. Chem., 173: 449, Roy, A. B.: The suiphatase of ox liver. I. The complex nature of the enzyme. Biochem. J., 53: 12, Gianetto, R. and De Duve, C.: Tissue fractionation studies. IV. Comparative study of the binding of acid phosphatase, p-glucuronidase and eathepsin by rat-liver particles. Biochem. J., 59: 433, De Duve, C. Pressman, B. C., Gianetto, R., Wattiaux,. and Appelmans, F.: Tissue fractionation studies. VI. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem. J., 60: 604, Dingle, J. T.: Studies on the mode of action of excess of vitamin A. III. Release of a. bound protease by the action of vitamin A.. Biochem. J., 79: 509, Lowry, 0. II., Rosebrough, Nira J., Farr, A.. L. and Randall, Rose J.: Protein measure-. ment with the folin phenol reagent. J. BioL Chem., 193: 265, Montagna, William: The Structure and Function of Skin, 2nd edition, 454 pp. New York,. Academic Press, Inc., Santoianni, P. and Rothman, S.: Nucleic acid splitting enzymes in human epidermis and! their possible role in keratinization. J. In vest. Derm., 37: 489, Wattiaux, R., Baudhuin, P., Berleur, Anne-- Marie and De Duve, C.: Tissue fractionation studies. VIII. Cellular localization of bound enzymes. Biochem. J., 63: 608, Koenig, H.: Histological distribution of brain gangliosides: Lysosomes as glycolipoprotein granules. Nature, London, 195: 782, Daems, W. Th. ind Van Rijssel, Th. G.: The fine structure of the peribiliary dense bodies in mouse liver tissue. J. Ultrastruct. Res., 5: 263, Koenig, H. and Jibril, A.: Acidic glycolipids and the role of ionic bonds in the structurelinked latency of lysosomal hydrolases. Biochim Biophys. Acta, 65: 543, Slater, T. F. and Riley, P. A.: Photosensitization and lysosomal damage. Nature, London, 209: 151, Hers, H. G.: Glycogen storage disease, Type II. In Whelan, W. J. and Cameron, Margaret P.: Ciba Foundation Symposium on Control of Glycogen Metabolism, pp Boston, Mass., Little, Brown & Company, Austin, James, McAfee, Donald, Armstrong, Donald, O'Rourke, Michael, Shearer, Leslie and Bachhawat, Bimal: Abnormal sulphatase activities in two human diseases (metachromatic leucodystrophy and gargoylism). Biochem. J., 93: 15c, Bean, M. A. and Janoff, A.: Lysosomal enzymes in Gaucher's disease. (Abstr.) Fed. Proc., 24: 617, 1965.